The ability of polypeptide ligands to bind cells and thereby elicit a phenotypic response such as cell growth, survival or differentiation is often mediated through transmembrane tyrosine kinases. The extracellular portion of each receptor tyrosine kinase (RTK) is generally the most distinctive portion of the molecule, as it provides the protein with its ligand-recognizing characteristic. Binding of a ligand to the extracellular domain results in signal transduction via an intracellular tyrosine kinase catalytic domain which transmits a biological signal to intracellular target proteins. The particular array of sequence motifs of this cytoplasmic, catalytic domain determines its access to potential kinase substrates (Mohammadi, et al., 1990, Mol. Cell. Biol., 11: 5068–5078; Fantl, et al., 1992, Cell, 69:413–413).
RTKs appear to undergo dimerization or some related conformational change following ligand binding (Schlessinger, J., 1988, Trend Biochem. Sci. 13:443–447; Ullrich and Schlessinger, 1990, Cell, 61:203–212; Schlessinger and Ullrich, 1992, Neuron 9:383–391); molecular interactions between dimerizing cytoplasmic domains lead to activation of kinase function. In some instances, such as the growth factor platelet derived growth factor (PDGF), the ligand is a dimer that binds two receptor molecules (Hart, et al., 1988, Science, 240: 1529–1531; Heldin, 1989, J. Biol. Chem. 264:8905–8912) while, for example, in the case of EGF, the ligand is a monomer (Weber, et al., 1984, J. Biol. Chem., 259:14631–14636).
Throughout the history of the biotechnology industry, many novel genes and associated proteins have been identified by virtue of their sequence homology with known genes. Many such proteins are purported to be receptors, but since their cognate ligands have not been identified, they are referred to as orphan receptors. The screening of many of these orphan receptors often leads to the identification of ligands that are capable of binding to the receptor, although the binding is often not associated with activation of any intracellular kinases or any other phenotypic change. Such was the case for members of the Eph receptor family. For sake of clarity, applicants incorporate by reference herein a letter cited as Eph Nomenclature Committee, 1997, published in Cell vol. 90: 403–403 (1997) which sets forth a nomenclature for the Eph Receptor and Eph Ligand Families.
Little, if any, biological activity had been observed in response to binding of a ligand to an Eph family receptor prior to the discovery as set forth in U.S. Pat. No. 5,747,033 issued May 5, 1998. That patent describes the concept of “clustering” whereby the soluble domains of ligands were combined to create multimers capable of activating the cognate receptors. Applicants have now extended the concept of clustering to additional ligands outside the Eph family, for example, the Tie-2 receptor ligands known as the angiopoietins, and have also discovered that this method for production of homogeneous forms of clustered ligands is broadly applicable to improve the affinity and/or increase the activity of a ligand as compared to the native form of the ligand.
Angiopoietin-1 (Ang) is one of two known ligands for the Tie-2 receptor and has been shown to be an agonist for Tie-2 (Davis, et al, 1996, Cell 87:1161–1169), whereas the second known ligand, angiopoietin-2, has been shown to be a naturally occurring antagonist of the Tie-2 receptor (Maisonpierre, et al., 1997, Science 277:55–60). Ang1* is a mutant form of angiopoietin-1 that comprises the N-terminal domain of angiopoietin-2 fused to the coiled-coil domain and the fibrinogen domain of angiopoietin-1 and that has a Cys to Ser mutation at amino acid 245. Ang1* has been shown to be a potent agonist for the Tie-2 receptor.
Experiments with mutants of angiopoietin-1 and angiopoietin-2 have demonstrated that the fibrinogen domains (FD) are the receptor-binding domains, and that dimerized versions of, for example Ang-1-FD-Fc, (i.e., the fibrinogen domain of Ang-1 fused to an Fc domain), can bind to the Tie-2 receptor with much higher affinity than monomeric Ang-1-FD (dimerization occurs due to the interaction between the Fc components of adjacent molecules). However, Ang-1-FD-Fc is not able to induce phosphorylation (activate) the Tie-2 receptor on endothelial cells unless it is further clustered with goat anti-human Fc antibodies (Jackson Immunoresearch). For this reason, mutant versions of Ang-1-FD and Ang-2-FD (i.e., the fibrinogen domain of Ang-2) were designed that were intrinsically more highly clustered.